Glioblastoma multiform (GBM) is the most common of all primary brain tumors with limited therapeutic options due to the diffusely infiltrative nature f the tumor and the presence of the blood brain barrier (BBB). Given an ability of T cells to penetrate the BBB and infiltrate tumors, T cells expressing chimeric antigen receptors (CARs) targeting tumor specific antigens have the potential to serve as a unique cytotoxic tool specifically targeting primary tumors or metastases that localize to the brain. IL13R?2, the high affinity IL13 receptor is a promising candidate for GBM-targeted therapy since it is expressed at a high frequency in GBM, but not in normal tissues. The majority of prior IL13R?2-targeted approaches take advantage of IL13 mutinies, which also recognize IL13R?1-positive target cells, raising concerns of 'on target/off cancer' toxicities. We have therefore developed a high affinity IL13R?2-specific monoclonal antibody (MAb) that does not cross-react with IL13R?1. Importantly, our preliminary results indicate that T cells expressing CARs that contain a single chain variable fragment (scFv) of IL13R?2-specific MAb (IL13R?2-CAR T cells), are specific for IL13R?2 and do not recognize or kill target cells expressing IL13R?1. In this proposal we now hypothesize that IL13R?2-CAR T cells will have potent anti-glioma activity in preclinical GBM models. CAR T cells have been primarily evaluated in xenograft models that lack salient features of GBMs; most importantly, they do not recapitulate their immunosuppressive tumor microenvironment, limiting preclinical testing and future development of CAR T cells. We hypothesize that these limitations can be overcome by adapting immune competent GBM models, which closely mimics human disease for in vivo testing of IL13R?2-CAR T cells. Our hypotheses will be evaluated in two interrelated Aims. In Aim 1 we will construct a panel of IL13R?2-CARs that contain our new scFvIL13R?2 as an ectodomain, a short or long hinge, a transmembrane domain, and endodomains that contain a CD3? signaling domain, and signaling domains derived from co-stimulatory molecules (CD28.?, CD28.CD134.?, or CD28.CD137. ?). The effector function of murine T cells expressing these CARs will be evaluated in vitro using standard immunological assays. While the results of our in vitro experiments will allow us to answer questions pertinent to optimal functioning of our experimental system, these data will not be fully representing the in vivo setting. To bridge this gap, we will compare in Aim 2 optimized IL13R?2-CARs in vivo using the GL261 and SMA-560 immune competent glioma models. We expect at the conclusion of the grant that we will have determined the optimal design of IL13R?2-CARs and have completed the testing of IL13R?2-CAR T cells in relevant, immune competent glioma models. While we use this data to justify the development of a Phase I clinical trial, the developed model will also be useful for future studies in which we aim to combine IL13R?2-CAR T cells with other glioma-targeted therapies.